The opportunistic pathogen P. aeruginosa remains a problem in the nosicomial infection of immunocompromised individuals. P. aeruginosa infections are particularly a problem in burn patients, people receiving medical implants, and in individuals suffering from cystic fibrosis (Fick, R. B. Jr., 1993). The organism is intrinsically resistant to many antibiotics and capable of forming biofilms which are recalcitrant to treatment. Several virulence factors have been identified in the pathogenesis of P. aeruginosa infections, including proteins such as exotoxin A, proteases, and exopolysaccharides including alginate and lipopolysaccharide (LPS). The LPS of P. aeruginosa is typical of Gram-negative bacteria, composed of lipid A-core oligosaccharide-O antigen repeating units.
P. aeruginosa is capable of coexpressing two distinct forms of LPS, designated A-band and B-band LPS, respectively. A-band LPS is a shorter, common form expressed by the majority of P. aeruginosa serotypes, and has a trisaccharide repeating unit of .alpha.-D-rhamnose linked 1.fwdarw.3, 1.fwdarw.3, 1.fwdarw.2. B-band LPS is the serotype-specific, O-antigen-containing form, and is a heteropolymer composed of di- to pentasaccharide repeats containing a wide variety of acyl sugars, amino sugars, and uronic acids. Both the A- and B-band repeating units are attached to lipid A-core, but there appear to be differences between them regarding point of attachment to and composition of the outer core region (Rivera et al., 1992).
The gene clusters for biosynthesis of core oligosaccharides/O-antigens rfb have been cloned and characterized from several bacterial species, including some from non-enteric genera such as Bordetella (Allen and Maskell, 1996), Haemophilus (Jarosik and Hansen, 1994), Neisseria (Gotschlich, 1994), Vibrio (Stroeher et al., 1992; Amor and Mutharia, 1995; Comstock et al., 1996), and Xanthamonas (Kingsley et al., 1993).
rfb clusters appear to be composed of mosaics of biosynthetic genes acquired horizontally from different sources (Reeves, 1993). Biochemical characterization of O-antigens from various species has shown that conservation of structure does not necessarily mirror conservation at the genetic level. Strains with identical O-antigens can differ significantly in their rfb clusters, while unique O-antigens can be encoded by only slightly variant rfb genes in other strains (Whitfield and Valvano, 1993).
Lightfoot and Lam were the first to report the cloning of genes involved in the expression of A-band (Lightfoot and Lam, 1991) and B-band (Lightfoot and Lam, 1993) LPS of P. aeruginosa. A recombinant cosmid clone pFV3 complemented A-band LPS synthesis in an A-band-deficient mutant, rd7513. pFV3 also mediated A-band LPS synthesis in five of the six P. aeruginosa O serotypes which lack A-band LPS. Another cosmid clone, pFV100, complemented B-band LPS synthesis in mutant ge6, which lacks B-band LPS. Physical mapping of the genes involved in A-band and B-band LPS synthesis indicated that the two gene clusters are physically distinct and are separated by more than 1.9 Mbp on the P. aeruginosa PAO1 genome. A-band LPS genes mapped between 5.75 and 5.89 Mbp (10.5 to 13.3 min), and B-band LPS genes mapped at 1.9 Mbp (near 37 min) on the 5.9-Mbp chromosome.
The structure of the P. aeruginosa O5 O-antigen has been elucidated (Knirel et al., 1988). O5 has a trisaccharide repeating unit of 2-acetamido-3-acetamidino-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-D-mannuronic acid, and N-acetyl-D-fucosamine (FIG. 30). Serotypes O2, O16, O18, and O20 of P. aeruginosa have similar O-antigens to serotype O5, varying only in one linkage or one epimer from O5 (Knirel et al., 1988) (FIG. 30). Immunochemical cross reactions have also been demonstrated among LPS of serotypes O2, O5 and O16 by the use of monoclonal antibodies (Lam et al., 1992). The rfbA (herein also referred to as "psbL" and "wbp1") from the O5 gene cluster has been characterized (Dasgupta and Lam, 1995). This O5 O-antigen biosynthetic gene has been shown to hybridize only with chromosomal DNA from the group of five serotypes with similar O-antigens, and not with the remaining fifteen serotypes.
There are currently three pathways proposed for biosynthesis and assembly of LPS, the Rfc-dependent and Rfcindependent pathways. Rfc is the O-antigen polymerase, and appears to be required for assembly of heteropolymeric O-antigens (Makela and Stocker, 1984). In contrast, homopolymeric O-antigens appear to be assembled without an O-antigen polymerase (Whitfield, 1995). Rfc-dependent (or Wzy) LPS synthesis has been shown to involve at least two other gene products which act in concert with Rfc; RfbX (or Wzx), the putative flippase which translocates individual O-antigen units across the cytoplasmic membrane where they are polymerized by Rfc (or Wzy), and Rol (or Wzz), the regulator of O-antigen chain length, which determines the preferred O-antigen chain length characteristic of the individual strain or serotype (Batchelor et al., 1993; Bastin et al., 1993; Morona et al., 1994b; Dodgson et al., 1996).